Prenatal exposure to ultrasound waves impacts neuronal migration in mice

Abstract

Neurons of the cerebral neocortex in mammals, including humans, are generated during fetal life in the proliferative zones and then migrate to their final destinations by following an inside-to-outside sequence. The present study examined the effect of ultrasound waves (USW) on neuronal position within the embryonic cerebral cortex in mice. We used a single BrdU injection to label neurons generated at embryonic day 16 and destined for the superficial cortical layers. Our analysis of over 335 animals reveals that, when exposed to USW for a total of 30 min or longer during the period of their migration, a small but statistically significant number of neurons fail to acquire their proper position and remain scattered within inappropriate cortical layers and/or in the subjacent white matter. The magnitude of dispersion of labeled neurons was variable but systematically increased with duration of exposure to USW. These results call for a further investigation in larger and slower-developing brains of non-human primates and continued scrutiny of unnecessarily long prenatal ultrasound exposure.

Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.

The experimental design of the system used for exposure of pregnant mice to USW. The mouse is placed in the tube chamber with abdomen embedded in conductant jelly and separated from the transducer by a tissue stand-off pad (TSP). Diagnostic levels of USW were delivered during 5–45 min. The embryos were monitored on the screen, and pulse, oxygenation, and temperature in the mothers were recorded (35).

Fig. 2.

Histological and immunohistochemical staining of sham and USW cortical slices. (A and B) Coronal sections across the cerebral wall in P10 control mouse (A) and mouse exposed to USW for 60 min between E17 and E18 (B). Quantification of BrdU-labeled cells was then performed (white boxes). (C–E) Control section from P10 controls animals. (F–H) Sections from P10 animals exposed as embryos to 60 min of USW. Neurons labeled with BrdU are stained green, and the slice was counterstained with propidium iodide, which is stained red. (E and H) The sections were divided into 10 equally spaced bins, with bin 1 starting at the pia and bin 10 ending near the ependymal surface. Notice the lower number of BrdU-labeled cells in the upper layers compared with the larger number in the deeper layers and subjacent white matter (white arrowheads). (Scale bars: B, 1.8 mm; G, 130 μm.)

Fig. 3.

Double-labeling of cortical slices exposed to USW for a cumulative dose of 30 min. (A–C) Staining for NeuN and BrdU and the merge of the two, respectively. The majority of BrdU+ cells are NeuN+, including those in layer 6 and subjacent white matter (white arrowheads in C). (D–F) No double-labeling for FoxP2, a marker for lower cortical layers, is present. Shown are BrdU+ cells in layer 6 and subjacent white matter. (G–I) Some BrdU+ cells in the subjacent white matter also label with Brn1, a marker of upper cortical layers (white arrowheads in I). (F and I) White arrows show the level of the ependyma in these images. (Scale bars: C, 72 μm; F and I, 65 μm.)

Fig. 4.

Percentage of cells in each bin averaged across all animals in each exposure duration, shown by condition. (A Left–F Left) Line graphs showing the percent of cells in each bin for experimental and control animals for exposure durations of 420, 210, 60, 30, 15, and 5 min, respectively. For durations >15 min, the proportion of cells in the lower five bins was significantly higher for exposed than for control animals. (A Right–F Right) Line graphs showing the ratio of the percentage of cells in ultrasound-exposed versus control animals (U/C) for each bin. For durations >15 min, the small differences in the absolute magnitudes in the lower bins represent large proportional increases. (G) (Left) The average percentage of cells in each bin for all 146 experimental animals and 141 sham controls across all durations. Note the excess of cells in the lower bins for the exposed animal. (Right) Line graph showing the ratio of the percentage of cells in ultrasound versus control animals for each bin. The small difference in the absolute magnitudes in the lower bins represents large proportional increases.

Fig. 5.

Dispersion of BrdU+ cells for USW and controls by duration of exposure. Dispersion is defined as the percentage of cells in bins 6–10. (A) The mean dispersion by condition and duration, with error bars showing the standard errors. (B) The mean dispersion for control and experimental animals across all durations with the Y-bars showing the standard errors. (C) A scatter plot by condition and duration for USW (red) and sham control (blue) conditions. Dispersion increases systematically with the length of exposure to USW. Dispersion also increases with sham exposure, although not as quickly as with ultrasound. (D) A scatter plot by condition with 0 min of duration for normal controls. The mean dispersion for the normal control condition was not significantly different from the 5-, 15-, 30-, 60-, and 210-min exposures. NC, normal control.

Fig. 6.

Schematic representation of the progression of neuronal migration to the superficial cortical layers in the normal mouse. (A–D) Most cells labeled with BrdU at E16 arrive in the cortex by E18, and, by P1, those cells become surpassed by subsequently generated neurons. Eventually, these cells will settle predominantly in layers 2 and 3 of the cerebrum. (E–H) Model of the USW effect. When cells generated at E16 are exposed to USW, they slow down on E17, and some remain in the white matter or are stacked in the deeper cortical layers.